Transgenic potato lines expressing hairpin RNAi construct of molting-associated EcR gene exhibit enhanced resistance against Colorado potato beetle (Leptinotarsa decemlineata, Say)
Most of the commercialized insect resistant transgenic crops express cry gene(s) isolated from Bacillus thuringiensis; however, intensive cultivation of Bt crops over almost two decades has been questioned regarding its sustainability and durability in pest management. The present study focused on silencing of highly specific molting-associated Ecdysone receptor (EcR) gene of Colorado potato beetle (CPB) using RNA interference (RNAi) approach. The partial cDNA of EcR gene of CPB was amplified using specific primers in sense and anti-sense orientations, and cloned in pRNAi-GG vector flanked by an intronic sequence (pdk). Leaf and internodal explants of Agria and Lady Olympia potato cultivars were infected with Agrobacterium strain LBA4404 harboring constructs under the control of CaMV 35S promoter. Standard molecular analysis of primary transformants showed proper integration of T-DNA in plant genome. The transgenic plants of both cultivars were evaluated for their efficacy against first, second and third instar CPB larvae. The leaf biotoxicity assays revealed 15–80% of CPB mortality. A significantly lower fold-change (0.87–4.14×) in larval weight was observed in insects fed on transgenic plants compared to the ones fed on control plants (1.87–6.53×). Furthermore, CPB larvae fed on transgenic plants exhibited reduced EcR transcripts, indicating the functionality of dsRNA EcR in silencing EcR gene expression. This study is an excellent example of the integration of an alternative, effective and reliable method to cope with potato insect pests that incur significant losses to potato production in the world.
KeywordsEcdysone receptor Transgenic technology Insect resistance Molting
The present research project was supported by “The Scientific and Technological Council of Turkey” (Tübitak, Project No. 215O520). The authors are highly thankful to Tübitak for providing PhD fellowship to Ms. Tahira Hussain from the project budget. Authors acknowledge the contributions of Prof. Dr. Ayhan GÖKÇE, Muhammad Saleem and Mr. Nadir Naqqash for allowing us to use facilities of entomology laboratory in Department of Plant Production and Technologies.
AB designed the study and secured funding for the project. TH constructed recombinant vectors, optimized transformation protocol for potato cultivars, conducted leaf bioassays. AB, EA and TH interpreted the results, and prepared the manuscript. AB and MEÇ supervised overall activities of the work.
Compliance with ethical standards
Conflict of interest
Authors declare no conflict of interest.
- Bakhsh A, Baloch FS, Hatipoğlu R, Özkan H (2015) Use of genetic engineering: benefits and health concerns. In: Hui YH, Evranuz EÖ (eds) Handbook of vegetable preservation and processing, 2nd edn. CRC Press, pp 100–131Google Scholar
- Douglas CJ, Staneloni RJ, Rubin RA, Nester EW (1985) Identification and genetic analysis of an Agrobacterium tumefaciens chromosomal virulence region. J Bacteriol 161:850–860Google Scholar
- FAOSTAT data (2017) http://www.fao.org/home/en/. Retrieved 30 July 2018
- Frary A, Earle ED (1996) An examination of factors affecting the efficiency of Agrobacterium-mediated transformation of tomato. Plant Cell Rep 16:235–240Google Scholar
- Sambrook J, Russell DW, Maniatis T (2001) Molecular cloning, vol 1-3. Cold Spring Habour Laboratory Press, New YorkGoogle Scholar
- Sehnal F (1989) Hormonal role of ecdysteroids in insect larvae and during metamorphosis. In: Koolman J (ed) Ecdysone. From chemistry to mode of action. Georg thieme Verlag, Stuttgart, pp 271–278Google Scholar
- Soto N, Enríquez GA, Ferreira A, Corrada M, Fuentes A, Tiel K, Pujol M (2007) Efficient transformation of potato stems segments from cultivar Désirée using phosphinothricin as selection marker. Biotecnol Apl 24:139–144Google Scholar
- Upadhyay SK, Chandrashekar K, Thakur N, Verma PC, Borgio JF, Singh PK, Tuli R (2011) RNA interference for the control of whiteflies (Bemisia tabaci) by oral route. J Bio 36:153–161Google Scholar